Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.
Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T-cell cytotoxicity. However, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules, as well as transmembrane and hinge domains have been added to form CARs of second and third generations, leading to some successful therapeutic trials in humans, where T-cells could be redirected against malignant cells expressing CD19 (June et al., 2011). However, the particular combination of signaling domains, transmembrane and co-stimulatory domains used with respect to CD19 ScFv, was rather antigen-specific and cannot be expanded to any antigen markers.
Acute myeloid leukaemia (AML) is the second most common acute leukaemia with approximately 13,300 new cases per year in the United States and 8,800 annual deaths. The commonly applied therapy of leukaemic diseases includes irradiation and/or chemotherapy. Furthermore, under certain circumstances, the additional possibility of bone marrow transplantation is regarded suitable. However, these therapies are relatively toxic to the patient and very often do not lead to a complete cure from the disease. Thus, although a complete remission can be achieved for 65-80% of patients receiving chemotherapy, most of these patients relapse (Cros et al., 2004) because the cells that survived the chemotherapy are enriched in AML leukaemia stem cells (AML-LSCs), and constitute a particularly dangerous reservoir of cells capable of re-expanding and causing a relapse. Leukaemia stem cells have been particularly well characterized for acute myeloid leukaemia. AML-LSCs express a characteristic set of cell-surface antigens including among others CD33. Patients older than 60 years have a poor prognosis with only 10% to 15% of 4-year disease-free survival (Gardin et al., 2007). This high relapse rate for AML patients and the poor prognosis for older patients highlight the urgent need for novel therapeutics preferentially targeting CD33+ cells.
CD33 (Sialic acid-binding Ig-like lectin 3) or SIGLEC3, referred to as P20138 under the UniProtKB/Swiss-Prot protein database, is a transmembrane receptor expressed on cells of myeloid lineage. It is usually considered myeloid-specific, but it can also be found on some lymphoid cells. It binds sialic acids, therefore is a member of the SIGLEC family of lectins.
In the past, different approaches have been used to develop unconjugated monoclonal antibodies with antitumor activity against CD33. However these attempts failed to address malignant cells specifically.
In 2000 Gemtuzumabozogamicin (Mylotarg™, GO), a calicheamicinconjugated humanized anti-CD33 monoclonal antibody, was approved by the American Food and Drug Administration (FDA) for treating patients older than 60 years with refractory or relapsed AML. However this was withdrawn from the market on Jun. 21, 2010; GO consisted of a humanized anti-CD33 IgG-antibody, chemically coupled to the cytotoxic agent calicheamicin. Post-approval study (SWOG 50106) raised concerns about the product's safety, while other clinical trials (British MRC AML-15 and the HOVON-43 trials) failed to demonstrate any clinical benefit (Maniecki et al., 2011). Side effects were found to include hepatic veno-occlusive disease, pulmonary toxicity and severe hypersensitivity reactions, whereas in vitro studies revealed antigen-independent cytotoxicities towards CD33 negative cell lines (Schwemmlein of al, 2006).
More recently, tri-specific polypeptide molecules combining immunoglobulin domains from CD123, CD16 and CD33 antibodies were proposed (WO2011/070109) to obviate the specificity issues previously encountered with therapeutics targeting CD33.
As an alternative to the previous strategies, the present invention provides with CD33 specific CARs, which can be expressed in immune cells to target CD33+ malignant cells with significant clinical advantage.